Vascular biosafety of commercial hydroxyapatite particles: discrepancy between blood compatibility assays and endothelial cell behavior.

BACKGROUND: Vascular homeostasis is ensured by a dynamic interplay involving the endothelium, the platelets and the coagulation system. Thus, the vascular safety of particulate materials must address this integrated system, an approach that has been largely neglected. This work analysed the effects of commercial hydroxyapatite (HA) particles in blood compatibility and in endothelial cell behavior, due to their clinical relevance and scarcity of data on their vascular biosafety. RESULTS: Particles with similar chemical composition and distinct size and morphology were tested, i.e. rod-like, nano dimensions and low aspect ratio (HAp1) and needle-shape with wider size and aspect ratio (HAp2). HAp1 and HAp2, at 1 to 10 mg/mL, did not affect haemolysis, platelet adhesion, aggregation and activation, or the coagulation system (intrinsic and extrinsic pathways), although HAp2 exhibited a slight thrombogenic potential at 10 mg/mL. Notwithstanding, significantly lower levels presented dose-dependent toxicity on endothelial cells' behavior. HAp1 and HAp2 decreased cell viability at levels ≥ 250 and ≥ 50 µg/mL, respectively. At 10 and 50 µg/mL, HAp1 did not interfere with the F-actin cytoskeleton, apoptotic index, cell cycle progression, expression of vWF, VECad and CD31, and the ability to form a network of tubular-like structures. Comparatively, HAp2 caused dose-dependent toxic effects in these parameters in the same concentration range. CONCLUSION: The most relevant observation is the great discrepancy of HA particles' levels that interfere with the routine blood compatibility assays and the endothelial cell behavior. Further, this difference was also found to be dependent on the particles' size, morphology and aspect ratio, emphasizing the need of a complementary biological characterization, taking into consideration the endothelial cells' functionality, to establish the vascular safety of particulate HA.

A highly reproducible continuous process for hydroxyapatite nanoparticles synthesis.

This work presents a continuous process for producing hydroxyapatite nanoparticles (NanoXIM) in a network reactor, NETmix, fed by a calcium solution, a phosphorus solution and an alkaline solution. Hydroxyapatite is considered a biomaterial, used as: food additives and nutritional supplements; bone graft for bone replacement, growth and repair; biocements and coating of metallic implant. Some of the most recent applications include their use in cosmetics, toothpaste and in esthetical treatments for diminishing wrinkles by stimulating conjunctive tissue formation. The proposed process enables the micromixing control, which is essential to form nanometric structures, but it is also a determining factor in the crystals purity, crystallinity and morphology. The reactants distribution scheme at the inlet of the reactor and along the reactor, performed continuously or varying in time, is also a crucial factor to programme the properties of reactant media along the reactor, such as the pH, the supersaturation degree, the Ca/P molar ratio, and the temperature. The calcium phosphate nanoparticles suspension that exits the reactor is submitted to further aging, separation, drying, sintering and milling processes.